Methods for determining gene function

1. Methods for determining gene function Study the effects of disrupting gene function (ap3 mutants) Examine the expression of the gene (GUS, In situ)

2. Methods for examining the expression of a gene Reporter construct (AP1::GUS) Pro- easy, can be used to define promoter region Con- indirect, requires transgenic plants In situ hybridization (today)

3. In Situ Hybridization • Molecular technique that allows one to detect localization of mRNA in a tissue • Pros • represents the best estimate of the actual expression pattern of a gene • does not require the production of transgenic plants • Cons • can be technically challenging • the expression of some genes is difficult to detect • Cannot be used to determine promoter region

4. Hypothetical block of plant tissue A mRNA mRNA of GeneX B C

5. mRNA of your favorite gene- a sense strand NBT/BCIP AP in situ probe Purple anti-DIG antibody DIG 3’ 5’ 5’ 3’ AAAAA-3’ 5’ mRNA- sense orientation

6. All cells that express your gene of interest turn purple Wild-type leaf tissue Gene X probe 05/08/06

7. All cells that express your gene of interest turn purple Wild-type leaf tissue Gene X probe 05/08/06

8. An example of AGAMOUS expression the an inflorescence